Real-time monitoring of intracellular Staphylococcus aureus replication

A high-throughput system to rapidly assess the intracellular replication of Staphylococcus aureus has been developed utilizing S. aureus transformed with a dual gfp-luxABCDE reporter operon under the control of a growth-dependent promoter. Replication of tagged bacteria internalized into bovine mammary epithelial cells (MAC-T) could be measured by monitoring fluorescence and bioluminescence from the reporter operon following removal of extracellular bacteria from the plates. Bacterial replication inside cells was confirmed by a novel ex vivo time-lapse confocal microscopic method. This assay of bacterial replication was used to evaluate the efficacy of antibiotics which are commonly used to treat staphylococcal infections. Not all antibiotics tested were able to prevent intracellular replication of S. aureus and some were ineffective at preventing replication of intracellular bacteria at concentrations above the MIC determined for bacteria in broth culture. Comparison of the fluorescence and bioluminescence signals from the bacteria enabled effects on protein synthesis and metabolism to be discriminated and gave information on the entry of compounds into the eukaryotic cell, even if bacterial replication was not prevented. Elevated resistance of S. aureus to antibiotics inside host cells increases the likelihood of selecting S. aureus strains which are resistant to commonly used antimicrobial agents within the intracellular niche. The approach presented directly assesses intracellular efficacy of antibiotics and provides an evidence-based approach to antibiotic selection for prescribing physicians and medical microbiologists.     

Intracellular ethanol accumulation in yeast cells during aerobic fermentation: a Raman spectroscopic exploration

Aims: To investigate the intracellular ethanol accumulation in yeast cells by using laser tweezers Raman spectroscopy (LTRS). Methods and Results: Ethanol accumulation in individual yeast cells during aerobic fermentation triggered by excess glucose was studied using LTRS. Its amount was obtained by comparing intracellular and extracellular ethanol concentrations during initial process of ethanol production. We found that (i) yeasts start to produce ethanol within 3 min after triggering aerobic fermentation, (ii) average ratio of intracellular to extracellular ethanol is 1·54 ± 0·17 during the initial 3 h after addition of 10% (w/v) excess glucose and (iii) the accumulated intracellular ethanol is released when aerobic fermentation is stimulated with decreasing glucose concentration. Conclusions: Intracellular ethanol accumulation occurs in initial stage of a rapid aerobic fermentation and high glucose concentration may attribute to this accumulation process. Significance and Impact of the Study: This work demonstrates LTRS is a real‐time, reagent‐free, in situ technique and a powerful tool to study kinetic process of ethanol fermentation. This work also provides further information on the intracellular ethanol accumulation in yeast cells.     

Incidence and possibilities of screening antifungal non-polyene antibiotics in actinomycetes

The author studied 114 strains of actinomycetes recovered from different geographical localities with different vegetation. Under submerse cultivation conditions 57% of the strains displayed antibiotic activity—48% antibacterial, 56% antifungal. As distinct from intracellular antibiotics, production of extracellular antibiotics was manifestly influenced by the composition of the fermentation medium. The commonest antifungal antibiotics were polyenes and non-polyenes, in the ratio 2∶1 Twenty strains produced non-polyene antifungal antibiotics, 14 of which corresponded to substances described in the literature. Only three strains produced antifungal non-polyenes singly; the others simultaneously formed basic antibacterial antibiotics of the type of streptomycin, streptothricin and neomycin or (also) polyene antibiotics, as a third component. Non-polyenes and polyenes did not occur together in the mycelium only or in fermentation fluid filtrate only. No correlation was found between the formation of non-polyenes and the place of origin of the strain. The possibility of screening new antibiotics is discussed.     

抗菌肽的基因工程研究进展

近年来细菌耐药性问题日趋严峻,寻找新型抗生素已迫在眉睫。抗菌肽是生物体产生的一种阳离子短肽,具有天然的抗菌活性。由于抗菌肽具有与传统抗生素不同的作用机制,不产生耐药性,因而具有重要的临床应用价值。但实践表明,抗菌肽的开发并非易事。针对近年来抗菌肽开发的基因工程策略和实践,尤其是大肠杆菌表达系统和酵母表达系统,进行了简要综述。     

Effect of Sorbinil on myo-Inositol Metabolism in Cultured Neuroblastoma Cells Exposed to Increased Glucose Levels

Neuroblastoma cells were used to determine the effect of sorbinil on myo-inositol metabolism in cells exposed to elevated levels of glucose in culture. Exposing cells to elevated levels of glucose led to an increase in levels of intracellular sorbitol. The increase in sorbitol levels was dependent on the extracellular glucose concentration. In contrast, the myo-inositol content of cells was decreased in the presence of increasing concentrations of extracellular glucose. Increasing the concentration of glucose in the culture medium caused a decrease in myo-inositol uptake and in the incorporation of extracellular myo-inositol into phospholipid. The effect of elevated glucose levels on myo-inositol metabolism and sorbitol accumulation was blocked by addition of 0.4 mM sorbinil. The ability of sorbinil to block the decrease in myo-inositol metabolism and sorbitol accumulation caused by 30 mM extracellular glucose was dependent on its concentration. Maximal effects were obtained with 0.4 mM sorbinil. However, there was some variation in the degree of effectiveness among batches of sorbinil. These results at the cellular level suggest that the intracellular accumulation of sorbitol is responsible for the alteration of myo-inositol metabolism observed in neuroblastoma cells exposed to elevated glucose concentrations.     

Reduction in adipocyte ATP by lipolytic agents: relation to intracellular free fatty acid accumulation

Epinephrine, norepinephrine, ACTH, and dibutyryl 3',5'-cyclic AMP reduced adipocyte ATP levels during 60 min incubation; glucose displayed a protective effect. The reduction in adipocyte ATP levels could not be attributed solely to: a direct hormone effect, deficiency in metabolic substrate, activation of adenyl cyclase with ATP consumption, loss of adenine nucleotide from the cell or loss of cells during incubation, lipolytic rate per se, or extracellular accumulation of FFA or glycerol. To determine whether intracellular FFA accumulation was a causative factor, intracellular FFA levels were measured during hormone-stimulated lipolysis. This was accomplished by using sucrose-U-(14)C as a marker for the extracellular space to correct for contamination of cells by extracellular albumin-bound FFA. These experiments showed that the fall in adipocyte ATP correlated with FFA saturation of medium albumin and progressive accumulation of FFA within the adipocyte. Furthermore, the protective effect of glucose noted above was associated with a marked reduction in intracellular FFA as compared to the extracellular FFA pool. On the basis of these studies, combined with those in the literature, it is concluded that in vitro effects of lipolytic agents on adipocyte ATP levels are the net result of imparied ATP synthesis (uncoupled oxidative phosphorylation) in the face of normal or augmented ATP consumption.     

The ability of an antimicrobial agent to penetrate a biofilm is not correlated with its killing or removal efficiency

The penetration ability of 12 antimicrobial agents, including antibiotics and biocides, was determined against biofilms of B. cereus and P. fluorescens using a colony biofilm assay. The surfactants benzalkonium chloride (BAC) and cetyltrimethyl ammonium bromide (CTAB), and the antibiotics ciprofloxacin and streptomycin were of interest due to their distinct activities. Erythromycin and CTAB were retarded by the presence of biofilms, whereas ciprofloxacin and BAC were not. The removal and killing efficacies of these four agents was additionally evaluated against biofilms formed in microtiter plates. The most efficient biocide was CTAB for both bacterial biofilms. Ciprofloxacin was the best antibiotic although none of the selected antimicrobial agents led to total biofilm removal and/or killing. Comparative analysis of the results obtained with colony biofilms and microtiter plate biofilms show that although extracellular polymeric substances and the biofilm structure are considered a determining factor in biofilm resistance, the ability of an antimicrobial agent to penetrate a biofilm is not correlated with its killing or removal efficiency. Also, the results reinforce the role of an appropriate antimicrobial selection as a key step in the design of disinfection processes for biofilm control.     

Antibiotic production by the marine photosynthetic bacterium Chromatium purpuratum NKPB 031704: localization of activity to the chromatophores

Abstract Over 200 strains of marine purple photosynthetic bacteria were isolated. Two strains showed antibiotic activity towards Saccharomyces cerevisiae and were tentatively identified as Chromatium purpuratum . Crude antibiotic, prepared by solvent extraction, showed a broad antimicrobial spectrum. The highest activity was found in the chromatophore fraction. Chromatographic separation of purified light harvesting complex from one strain, NKPB 031704, showed the presence of two separate pigmented compounds which were responsible for antimicrobial activity. Our findings reveal the unexpected ability of photosynthetic bacteria to produce broad spectrum antibiotics. In addition, this is the first example of intracellular localization of antibiotic activity in a marine bacterium.     

Impaired glucose transporter-1 degradation and increased glucose transport and oxidative stress in response to high glucose in chondrocytes from osteoarthritic versus normal human cartilage

Introduction  

Disorders that affect glucose metabolism, namely diabetes mellitus (DM), may favor the development and/or progression of osteoarthritis (OA). Thus far, little is known regarding the ability of chondrocytes to adjust to variations in the extracellular glucose concentration, resulting from hypoglycemia and hyperglycemia episodes, and so, to avoid deleterious effects resulting from deprivation or intracellular accumulation of glucose. The aim of this study was to compare the ability of normal and OA chondrocytes to regulate their glucose transport capacity in conditions of insufficient or excessive extracellular glucose and to identify the mechanisms involved and eventual deleterious consequences, namely the production of reactive oxygen species (ROS).     

Stimulation of the insulin secretory mechanism following barium accumulation in pancreatic beta-cells

Electrothermal atomic absorption spectroscopy was employed for measuring barium in beta-cell-rich pancreatic islets microdissected from ob/ob-mice. Both the uptake and efflux of barium displayed two distinct phases. There was a 4-fold accumulation of barium into intracellular stores when its extracellular concentration was 0.26 mM. Unlike divalent cations with more extensive intracellular accumulation, the washout of Ba2+ was not inhibited by D-glucose. Ba2+ served as a substitute for Ca2+ both in maintaining the glucose metabolism after removal of extracellular Ca2+ and making it possible for glucose to stimulate insulin release. Furthermore, Ba2+ elicited insulin release in the absence of glucose and other secretagogues. The latter effect was reversible and was markedly potentiated under conditions known to increase the beta-cell content of cyclic AMP. It is likely that the observed actions of Ba2+ are mediated by Ca2+, since Ca2+ -dependent regulatory proteins, such as calmodulin, apparently cannot bind Ba2+ specifically.     

Proline-rich antimicrobial peptides: potential therapeutics against antibiotic-resistant bacteria

The increasing resistance of pathogens to antibiotics causes a huge clinical burden that places great demands on academic researchers and the pharmaceutical industry for resolution. Antimicrobial peptides, part of native host defense, have emerged as novel potential antibiotic alternatives. Among the different classes of antimicrobial peptides, proline-rich antimicrobial peptides, predominantly sourced from insects, have been extensively investigated to study their specific modes of action. In this review, we focus on recent developments in these peptides. They show a variety of modes of actions, including mechanism shift at high concentration, non-lytic mechanisms, as well as possessing different intracellular targets and lipopolysaccharide binding activity. Furthermore, proline-rich antimicrobial peptides display the ability to not only modulate the immune system via cytokine activity or angiogenesis but also possess properties of penetrating cell membranes and crossing the blood brain barrier suggesting a role as potential novel carriers. Ongoing studies of these peptides will likely lead to the development of more potent antimicrobial peptides that may serve as important additions to the armoury of agents against bacterial infection and drug delivery.     

The Lysine Acetylation Modification in the Porin Aha1 of Aeromonas hydrophila Regulates the Uptake of Multidrug Antibiotics

Protein lysine acetylation (Kac) modification plays important roles in diverse physiological functions. However, there is little evidence on the role of Kac modification in bacterial antibiotic resistance. Here, we compared the differential expressions of whole-cell proteins and Kac peptides in oxytetracycline sensitive and oxytetracycline resistance (OXY^R) strains of Aeromonas hydrophila using quantitative proteomics technologies. We observed a porin family protein Aha1 downregulated in the OXY^R strain, which may have an important role in the OXY resistance. Interestingly, seven of eight Kac peptides of Aha1 decreased abundance in OXY^R as well. Microbiologic assays showed that the K57R, K187R, and K197R Aha1 mutants significantly increased antibiotic resistance to OXY and reduced the intracellular OXY accumulation in OXY stress. Moreover, these Aha1 mutants displayed multidrug resistance features to tetracyclines and β-lactam antibiotics. The 3D model prediction showed that the Kac states of K57, K187, and K197 sites located at the extracellular pore vestibule of Aha1 may be involved in the uptake of specific types of antibiotics. Overall, our results indicate a novel antibiotic resistance mechanism mediated by Kac modification, which may provide a clue for the development of antibiotic therapy strategies.     

Drug detoxification dynamics explain the postantibiotic effect

The postantibiotic effect (PAE) refers to the temporary suppression of bacterial growth following transient antibiotic treatment. This effect has been observed for decades for a wide variety of antibiotics and microbial species. However, despite empirical observations, a mechanistic understanding of this phenomenon is lacking. Using a combination of modeling and quantitative experiments, we show that the PAE can be explained by the temporal dynamics of drug detoxification in individual cells after an antibiotic is removed from the extracellular environment. These dynamics are dictated by both the export of the antibiotic and the intracellular titration of the antibiotic by its target. This mechanism is generally applicable for antibiotics with different modes of action. We further show that efflux inhibition is effective against certain antibiotic motifs, which may help explain mixed cotreatment success.     

Transport of monosaccharides in kidney-cortex cells

1. The aerobic transport of d-glucose and d-galactose in rabbit kidney tissue at 25 degrees was studied. 2. In slices forming glucose from added substrates an accumulation of glucose against its concentration gradient was found. The apparent ratio of intracellular ([S](i)) and extracellular ([S](o)) glucose concentrations was increased by 0.4mm-phlorrhizin and 0.3mm-ouabain. 3. Slices and isolated renal tubules actively accumulated glucose from the saline; the apparent [S](i)/[S](o) fell below 1.0 only at [S](o) higher than 0.5mm. 4. The rate of glucose oxidation by slices was characterized by the following parameters: K(m) 1.16mm; V(max.) 4.5mumoles/g. wet wt./hr. 5. The active accumulation of glucose from the saline was decreased by 0.1mm-2,4-dinitrophenol, 0.4mm-phlorrhizin and by the absence of external Na(+). 6. The kinetic parameters of galactose entry into the cells were: K(m) 1.5mm; V(max) 10mumoles/g. wet wt./hr. 7. The efflux kinetics from slices indicated two intracellular compartments for d-galactose. The galactose efflux was greatly diminished at 0 degrees , was inhibited by 0.4mm-phlorrhizin, but was insensitive to ouabain. 8. The following mechanism of glucose and galactose transport in renal tubular cells is suggested: (a) at the tubular membrane, these sugars are actively transported into the cells by a metabolically- and Na(+)-dependent phlorrhizin-sensitive mechanism; (b) at the basal cell membrane, these sugars are transported in accordance with their concentration gradient by a phlorrhizin-sensitive Na(+)-independent facilitated diffusion. The steady-state intracellular sugar concentration is determined by the kinetic parameters of active entry, passive outflow and intracellular utilization.     

Degradation of chicken feathers by Chrysosporium georgiae

Using a baiting technique, Chrysosporium georgiae was isolated from chicken feathers. Twenty-eight different fungal isolates were evaluated for their ability to produce keratinase enzymes using a keratin–salt agar medium containing either white chicken feathers or a prepared feather keratin suspension (KS). The Chrysosporium species were able to use keratin and grow at different rates. Chrysosporium georgiae completely degraded the added keratin after 9 days of incubation. Degradation of feathers by C. georgiae was affected by several cultural factors. Highest keratinolytic activity occurred after 3 weeks of incubation at 6 and 8~pH at 30 °C. Chrysosporium georgiae was able to degrade white chicken feathers, whereas bovine and human hair and sheep wool were not degraded and did not support fungal growth. Addition of 1% glucose to the medium containing keratin improved fungal growth and increased enzyme production. Higher keratin degradation resulted in high SH accumulation and the utilization of the carbohydrate carbon in the medium resulted in high keto-acid accumulation but decreased ammonia accumulation. Supplementation of the keratin–salt medium with minerals such as NH4Cl and MgSO4 slightly increased mycelial growth, but decreased production of extracelluar keratinase. Keratinase enzymes were very poorly produced in the absence of keratin, indicating its inducible nature. Analysis of endocellular keratinases in the mycelial homogenate indicated higher activity of intracellular keratinase as compared to the extracellular enzyme in culture filtrates. Chrysosporium georgiae was the most superior for keratinase production among the Chrysosporium species tested in the presence or absence of glucose. It produced more of the intracellular enzymes than the exocellular ones. This revised version was published online in June 2006 with corrections to the Cover Date.     

Compounds of Alpinia katsumadai as potential efflux inhibitors in Mycobacterium smegmatis

Efflux pumps are one of the well established mechanisms that contribute to antibiotic resistance in bacteria, such as mycobacteria. As a result, the identification of efflux pump inhibitors is an attractive target in antimicrobial therapy. The isolated compounds, three diarylheptanoids, trans,trans-1,7-diphenylhepta-4,6-dien-3-one (1), (5R)-trans-1,7-diphenyl-5-hydroxyhept-6-en-3-one (2), (3S,5S)-trans-1,7-diphenylhept-1-ene-3,5-diol (3) and the flavonoid pinocembrin (4), from Alpinia katsumadai, Zingiberaceae, were examined for their antimycobacterial activity and their synergistic effects with different antibiotics against M. smegmatis mc(2) 155. Furthermore, these compounds were evaluated as potential EtBr efflux inhibitors. Although they showed weak antimycobacterial activities (MIC ≥ 64 mg/L), especially compound 1 revealed a significant activity on the EtBr accumulation and efflux as well as a synergistic effect in combination with rifampicin.     

Metabolic constraints on the evolution of antibiotic resistance

Despite our continuous improvement in understanding antibiotic resistance, the interplay between natural selection of resistance mutations and the environment remains unclear. To investigate the role of bacterial metabolism in constraining the evolution of antibiotic resistance, we evolved Escherichia coli growing on glycolytic or gluconeogenic carbon sources to the selective pressure of three different antibiotics. Profiling more than 500 intracellular and extracellular putative metabolites in 190 evolved populations revealed that carbon and energy metabolism strongly constrained the evolutionary trajectories, both in terms of speed and mode of resistance acquisition. To interpret and explore the space of metabolome changes, we developed a novel constraint‐based modeling approach using the concept of shadow prices. This analysis, together with genome resequencing of resistant populations, identified condition‐dependent compensatory mechanisms of antibiotic resistance, such as the shift from respiratory to fermentative metabolism of glucose upon overexpression of efflux pumps. Moreover, metabolome‐based predictions revealed emerging weaknesses in resistant strains, such as the hypersensitivity to fosfomycin of ampicillin‐resistant strains. Overall, resolving metabolic adaptation throughout antibiotic‐driven evolutionary trajectories opens new perspectives in the fight against emerging antibiotic resistance.     

Glycolysis Controls Plasma Membrane Glucose Sensors To Promote Glucose Signaling in Yeasts

Sensing of extracellular glucose is necessary for cells to adapt to glucose variation in their environment. In the respiratory yeast Kluyveromyces lactis, extracellular glucose controls the expression of major glucose permease gene RAG1 through a cascade similar to the Saccharomyces cerevisiae Snf3/Rgt2/Rgt1 glucose signaling pathway. This regulation depends also on intracellular glucose metabolism since we previously showed that glucose induction of the RAG1 gene is abolished in glycolytic mutants. Here we show that glycolysis regulates RAG1 expression through the K. lactis Rgt1 (KlRgt1) glucose signaling pathway by targeting the localization and probably the stability of Rag4, the single Snf3/Rgt2-type glucose sensor of K. lactis. Additionally, the control exerted by glycolysis on glucose signaling seems to be conserved in S. cerevisiae. This retrocontrol might prevent yeasts from unnecessary glucose transport and intracellular glucose accumulation.     

Change of extracellular cAMP concentration is a sensitive reporter for bacterial fitness in high-cell-density cultures of Escherichia coli

Guanosine-3',5'-tetraphosphate (ppGpp) and sigmaS, two regulators of the starvation response of Escherichia coli, have received increasing attention for monitoring cell physiological changes in production processes, although both are difficult to quantify. The kinetics of cAMP formation and degradation were not yet investigated in such processes, although the complex regulation of cAMP by synthesis, release, and degradation in connection with straightforward methods for analysis renders it a highly informative target. Therefore, we followed the cAMP concentration in various nonrecombinant and in four different recombinant glucose-limited fed-batch processes in different production scales. The intracellular cAMP concentration increases strongly at the end of the batch phase. Most cAMP is released to the cultivation medium. The rates of accumulation and degradation of extracellular cAMP are growth-rate-dependent and show a distinct maximum at a growth rate of about 0.35 h(-1). At very low growth rates, below 0.05 h(-1), extracellular cAMP is not produced but rather degraded, independent of whether this low growth rate is caused by glucose limitation or by the high metabolic load of recombinant protein production. In contrast to intracellular cAMP, which is highly unstable, analysis of extracellular cAMP is simpler and the kinetics of accumulation and degradation reflect well the physiological situation, including unlimited growth, limitation, and severe starvation of a production host.     

辣椒溶杆菌X2-3 LC_Clp转录因子功能分析

辣椒溶杆菌(Lysobacter capsici) X2-3是从小麦根际分离的一株对多种病原真菌和卵菌有抑菌活性的菌株,目前对该菌株产生的抗菌物质及其产生调控机制尚不明确。【目的】明确转录因子LC_Clp对该菌株抗菌物质产生的调节作用,为深入了解L. capsici X2-3的生防机制提供依据。【方法】从转座子EZ-Tn5随机插入突变体库中筛选获得X2-3的LC_clp基因突变体M356,通过恢复性克隆获得功能互补菌株,分析LC_clp基因在拮抗活性、胞外酶分泌以及调控基因表达方面的差异。【结果】与X2-3相比,M356对测试病原真菌、卵菌的抑菌活性和产生体外抗菌物质的能力完全丧失,蛋白酶和纤维素酶产生量明显减少,几乎不产几丁质酶;所检测的转录调节因子、次生代谢及胞外酶等相关基因的表达量均显著低于野生株X2-3,而互补菌株MCS28和X2-3水平相当。【结论】LC_Clp不仅与菌株的抗菌物质合成及抑菌活性有关,还影响胞外酶的产生,并调控多种基因的表达,具有广泛的调节作用。